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Plasmodium falciparum: Genetic and immunogenic characterisation of the rhoptry neck protein PfRON4
Experimental Parasitology 122 (2009) 280–288
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Plasmodium falciparum: Genetic and immunogenic characterisation of the rhoptry neck protein PfRON4 Belinda J. Morahan a,1, Georgina B. Sallmann a, Robert Huestis a,b, Valentina Dubljevic a, Karena L. Waller a,* a b
Department of Microbiology, Monash University, Clayton, Vic. 3800, Australia Victorian Bioinformatics Consortium, Monash University, Clayton, Vic. 3800, Australia
a r t i c l e
i n f o
Article history: Received 19 January 2009 Received in revised form 7 April 2009 Accepted 29 April 2009 Available online 12 May 2009 Keywords: PfRON4 Malaria Plasmodium falciparum Rhoptry
a b s t r a c t The Apicomplexan parasites Toxoplasma and Plasmodium, respectively, cause toxoplasmosis and malaria in humans and although they invade different host cells they share largely conserved invasion mechanisms. Plasmodium falciparum merozoite invasion of red blood cells results from a series of co-ordinated events that comprise attachment of the merozoite, its re-orientation, release of the contents of the invasion-related apical organelles (the rhoptries and micronemes) followed by active propulsion of the merozoite into the cell via an actin-myosin motor. During this process, a tight junction between the parasite and red blood cell plasma membranes is formed and recent studies have identified rhoptry neck proteins, including PfRON4, that are specifically associated with the tight junction during invasion. Here, we report the structure of the gene that encodes PfRON4 and its apparent limited diversity amongst geographically diverse P. falciparum isolates. We also report that PfRON4 protein sequences elicit immunogenic responses in natural human malaria infections. Ó 2009 Elsevier Inc. All rights reserved.
1. Introduction The obligate intracellular protozoan parasites Toxoplasma and Plasmodium are both members of the Apicomplexan family and are the respective causative agents of the human parasitic diseases toxoplasmosis and malaria. Common to the invasive forms of all Apicomplexan parasites are a set of apically located secretory organelles, the rhoptries, micronemes and dense granules, which secrete numerous proteins, some of which are critically required for invasion of the target host cell. Although Toxoplasma and Plasmodium ultimately invade different cell types, they share a commonality in invasion mechanisms. For both parasites, invasion results from a series of co-ordinated events that comprise attachment of the invasive parasite to the host cell, its re-orientation and release of the contents of the apical organelles (rhoptries and micronemes) followed by active propulsion of the parasite into the host cell via an actin-myosin motor. During invasion, the irreAbbreviations: TgRON4, Toxoplasma gondii rhoptry neck protein 4; PfAMA1, Plasmodium falciparum apical membrane antigen 1; PfRON4, Plasmodium falciparum rhoptry neck protein 4; IFA, indirect immunofluorescence assay; GST, glutathione Stransferase; DAPI, 40 ,6-diamidino-2-phenylinodole dihydrochloride; PBS, phosphate buffered saline. * Corresponding author. Fax: +61 3 9902 9222. E-mail address: [email protected] (K.L. Waller). 1 Present address: Department of Biology, Loyola University, Chicago IL, USA. 0014-4894/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2009.04.013
versible juncture formed between the plasma membranes of the invading parasite and the host cell is called the tight junction, and results from a series of protein interactions between parasite ligands and their specific receptor molecules on the host cell surface. As invasion proceeds, the tight junction translocates along the sides of the invading parasite until the invasion process eventually terminates when the parasite is fully engulfed within a parasitophorous vacuole in the host cell (see Cowman and Crabb for recent review (2006)). Studies in Toxoplasma gondii have begun to tease apart the parasite proteins associated with the tight junction during invasion of host cells, one of which is TgRON4. TgRON4 appears to be refractory to genetic deletion and localises to the neck region of the rhoptries in non-invading tachyzoites, but in invading tachyzoites TgRON4 is secreted from the rhoptries and localises with the tight junction as it translocates along the sides of the invading parasite (Alexander et al., 2005; Lebrun et al., 2005). TgRON4 has also been shown to form a protein complex consisting of two other rhoptry neck proteins, TgRON2 and TgRON5, as well as the microneme-secreted and proteolytically processed integral membrane protein that is critically required for invasion, T. gondii Apical Membrane Antigen 1 (TgAMA1) (Alexander et al., 2005; Mital et al., 2005). Highlighting the significant level of conservation in parasite invasion mechanisms between T. gondii and P. falciparum, the P. falciparum homologues of each TgRON have recently been identified
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(Alexander et al., 2006; Bradley et al., 2005). PfRON4 has been shown to localise to the neck region of rhoptries in merozoites (Alexander et al., 2006) and is similarly found in complex with the leading malaria vaccine candidate PfAMA1 (Alexander et al., 2006), PfRON2 and PfRON5 (Cao et al., 2008; Collins et al., 2009). For both T. gondii and P. falciparum, the precise function of the RON4–AMA1 protein complex has yet to be delineated. In the P. falciparum genome database (www.plasmodb.org), the gene that encodes PfRON4 is incorrectly annotated. Here, we describe the correct structure of the gene that encodes PfRON4 and probe the level of conservation of PfRON4 sequences derived from geographically diverse P. falciparum isolates. We also investigate the immunogenicity of regions of PfRON4 with hyperimmune sera obtained from individuals living in malaria endemic regions of the world.
Homologous DNA sequences to pfron4 were extracted using BLAST searches from the genome database that is available online at the Broad Institute (http://www.broad.mit.edu/). Complete pfron4 nucleotide sequences were extracted for the P. falciparum isolates HB3 (Honduras; Walliker et al., 1987) and Dd2 (Indochina; Wellems et al., 1990). The pfron4 sequence for 3D7 (a clonal line derived from NF54 and of presumed African origin; (Ponnudurai et al., 1982) was derived by our manual re-annotation of sequence available from http://www.plasmodb.org. ClustalW nucleotide and protein sequence alignments were performed using software available through the European Bioinformatics Institute website (http:// www.ebi.ac.uk/Tools/clustalw/). 2.3. GST-PfRON4 fusion protein expression and purification
Plasmodium falciparum 3D7 parasites were maintained by in vitro culture using standard culture conditions (Trager and Jensen, 1976) and supplemented with 0.5% (w/v) AlbumaxII (Invitrogen). Parasitaemias were determined by microscopic examination of Giemsa stained thin blood smears. Highly synchronised parasite cultures were obtained by sorbitol synchronisation methods (Lambros and Vanderberg, 1979).
Recombinant glutathione S-transferase (GST) fusions proteins of regions of PfRON4 were generated. Six overlapping regions of pfron4, referred to as F1 through F6 (Fig. 3A), were amplified by PCR using specific primers (Table 1) and P. falciparum 3D7 cDNA as the template. The resultant DNA fragments were cloned into pGEX-KG (Guan and Dixon, 1991) and the encoded GST fusion proteins were expressed and purified using standard affinity chromatography (Bennett et al., 1997). The majority of the fusion proteins generated, excepting fragments F1 and F3, possessed a small overlap of 69 residues at the N- or C-terminus of each PfRON4 protein sequence resulting from overlap of the amplifying adjacent primer sequences.
2.2. PCR amplification, cloning and sequence analysis of pfron4
2.4. Antisera production and immunoblotting
Specific oligonucleotide primers (Table 1) were used to amplify full length pfron4 from P. falciparum 3D7 cDNA. Manual bioinformatic analyses performed by Robert Huestis (Victorian Bioinformatics Consortium) of the PF11_0168 nucleotide sequence (www.plasmodb.org) indicated it most likely contained an incorrect gene annotation. Our re-annotated three exon pfron4 gene structure (Fig. 1A) was confirmed by RT-PCR (Superscript II Kit, Invitrogen) using total RNA extracted with TrizolÒ (Invitrogen) from asynchronous parasite cultures. RT-PCR products were amplified using the Expand High Fidelity PCR System (Roche) from samples treated with or without Reverse Transcriptase (RT+ and RT, respectively). The amplified RT-PCR products were resolved by agarose gel electrophoresis in 1% (w/v) gels and then cloned using the pTOPO TA Cloning Kit for Sequencing (Invitrogen). Both the sense and antisense nucleotide sequences of two independent pfron4 cDNA clones were determined. Sequence analyses and alignments were performed using SeqManTM II software (DNAStar). The re-annotated and RT-PCR confirmed gene sequence of pfron4 has been reported to www.plasmodb.org.
Rabbit polyclonal antibodies were generated against the GSTPfRON4 fusion proteins F1 (amino acid residues 42–400; Proellocks et al., 2009) and F2 (amino acid residues 583–767), using previously described methods (Proellocks et al., 2009). Purified recombinant proteins and parasite lysates were resolved by denaturing SDS–PAGE in 12% (w/v) polyacrylamide gels and transferred to polyvinylidene fluoride (PVDF) membranes (NEN). Immunoblots were probed with various primary antibodies and detected using Western Lightning Chemiluminescence Reagent Plus (PerkinElmer Life Sciences). The primary antibodies used were polyclonal rabbit anti-PfRON4 F1 and anti-PfRON4 F2 (1/500), anti-GRP(BiP) antisera (1/500; Kumar et al., 1991), rabbit anti-GST (1/500 dilution) and pooled human hyperimmune sera (1/1000) obtained from malaria endemic regions of Vietnam (Wang et al., 2001) and Papua New Guinea (Marshall et al., 1997). Pooled hyperimmune sera samples consist of pooled equal volumes of at least 18 individual sera. Human control sera were obtained from individuals living in Melbourne, Australia that had not previously been exposed to malaria.
2. Materials and methods 2.1. Parasite culture
Table 1 Oligonucleotide primers. Primer
Sequence (50 ? 30 )a
Targetb
p1 p2 p3 p4 p5 p6 p7 p8 p9 p10 p11
ccggaattctaATGTCTAGTGTTAGATTTTTTTTATG cccaagcttTTATAAATCATCAAAAATCATCTTTTC ccggaattctaAGCCATATAGAAGAACCTCAA cccaagctATGTGAATGATGATTTATATTATTAT ccggaattctaTTTCCAAACGAGGATGATAATC cccaagcttTCTTTCTCTTAAAGATACATGCA ccggaattctaAATAATATAAATCATCATTCACATG cccaagcttATGATTATCATCCTCGTTTGG ccggaattctaTTGCATGTATCTTTAAGAGAAAG cccaagcttTGCTCCATATTCATCAGGAGC ccggaattcGGTTAGCTCCTGATGAATATGG
pfron4 pfron4 pfron4 pfron4 pfron4 pfron4 pfron4 pfron4 pfron4 pfron4 pfron4
a b
Sequences shown in upper case are gene specific. Underlined sequences indicate enzyme restriction sites. + and indicate sense and anti-sense strand sequences, respectively.
full length (+) and pfron4 F3 (+) full length () and pfron4 F6 () F1 (+) F1 () and pfron4 F3 () F2 (+) F2 () F4 (+) F4 () F5 (+) F5 () F6 (+)
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Fig. 1. Genomic structure of pfron4. (A) Schematic representation of the annotated, predicted and confirmed pfron4 genomic locus (exon sequences are drawn to scale). The gene sequence PF11_0168 (www.plasmodb.org) was reported as encoding PfRON4 (Alexander et al., 2005; Lebrun et al., 2005), and as such, is annotated to possess three exons in a tandem array; exon one (5471 bp) is located 999 bp upstream of exon 2 (81 bp), and exon 3 (3346 bp) is located a further 201 bp downstream. Closer examination of the genomic nucleotide sequence data indicated a likely mis-annotation. Manual re-annotation predicted that PfRON4 was instead encoded by a gene possessing three exons, in which the first exon (76 bp) was located 201 bp upstream of the second exon (3340 bp) with the third exon (190 bp) being located a further 129 bp downstream. In this prediction, the larger 5471 bp exon was excluded from the PfRON4 coding sequence. The relative locations of oligonucleotide primers used in RT-PCR are shown, along with the expected sizes of the amplified DNA products. (B) Confirmation of the predicted pfron4 genomic structure by RT-PCR. An approximately 3.6 kb cDNA fragment (indicated by the asterisk) was amplified from samples treated with Reverse Transcriptase (RT+) but not in non-treated samples (RT).
2.5. Indirect immunofluorescence assay (IFA) Thin blood smears were prepared from cultures rich in schizont stage parasites and fixed with acetone/methanol (9:1). IFAs were preformed using previously described methods (Waller et al., 2008). Cells were visualized by wide field fluorescent microscopy using an Olympus BX51 system microscope and an Olympus DP70 microscope digital camera. Images were captured using DP Controller and DPManager software (Olympus Optical Co.). The primary antibodies were: rabbit anti-PfRON4 F2 (1/500), rabbit anti-RAMA Fragment D (1/500; Topolska et al., 2004), mouse anti-Pf34 Fragment C (1/500; Proellocks et al., 2007), rat anti-EBA-175 region VI (1/500; Sim et al., 1990) and monoclonal anti-PfAMA1 1F9 (1/500; Coley et al., 2001). The secondary antibodies were: goat anti-rabbit, goat anti-rat and goat anti-mouse AlexaFluorÒ488 (Green) or AlexaFluorÒ568 (Red) conjugate antibodies (1/500; Molecular Probes). 2.6. Immunoprecipitation Immunoprecipitations of parasite material were performed using a methodology adapted from Cooke et al. (2006). Immunoprecipitated protein complexes were resolved by denaturing SDS–PAGE in 12% (v/v) acrylamide gels before immunoblotting with monoclonal anti-PfAMA1 1F9 antibody (1/1000; Coley et al., 2001) or mono-
clonal anti-PfRON4 antibody (1/500; Baum et al., 2008; Richard et al., 2009).
3. Results 3.1. pfron4 gene structure and sequence Previous reports have identified PF11_0168 as the gene sequence that encodes the rhoptry neck protein PfRON4 (Alexander et al., 2005; Lebrun et al., 2005). PF11_0168, as originally annotated in the P. falciparum genome database (www.plasmodb.org), consisted of three exons in a tandem array (Fig. 1A). Manual bioinformatic analysis of the putative intron/ exon boundaries proposed in this PF11_0168 sequence indicated that the sequence had most likely been incorrectly annotated. Manual re-annotation of this genomic locus suggested that although PfRON4 was indeed encoded by a gene possessing three exons in a tandem array, it appeared to exclude the larger 5471 bp sequence and instead include a previously unannotated downstream exon. Thus, we hypothesised that pfron4 was encoded by a first exon (76 bp) located 201 bp upstream of the second exon (3340 bp) with the third exon (190 bp) being located a further 129 bp downstream (Fig. 1A).
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In order to confirm that our manually re-annotated gene sequence encodes PfRON4, we performed RT-PCR on total RNA extracted from asynchronous parasite cultures. Specific primers (Table 1) were designed with reference to our predicted gene annotation to amplify the full length cDNA, which if our predicted exon model was correct, was expected to result in a 3606 bp spliced cDNA RT-PCR product (Fig. 1A). An approximately 3.6 kb product was successfully amplified by RT-PCR in RT+ samples. No amplification products were observed in RT- samples (Fig. 1B). The complete nucleotide sequences of two independent cloned pfron4 cDNAs were determined and compared to the reported sequence of PF11_0168 from the genome database (www.plasmodb.org). These comparisons demonstrated significant homology (data not shown) to the P. falciparum genome database’s PF11_0168 exon 2 and exon 3 sequences, but not to exon 1 (5471 bp exon; Fig. 1A). Instead, the nucleotide sequence obtained from the 30 end of both the cloned cDNA products was homologous to a previously unannotated exon located downstream of PF11_0168 exon 3. Thus, we have shown that PfRON4 is encoded by a three exon gene (Fig. 1A) that is only partially encoded by the sequence reported as PF11_0168 in the genome database (www.plasmodb.org). ClustalW alignments of the pfron4 sequence data extracted from the Broad Institute genome database (http://www.broad.mit.edu/) or www.plasmodb.org were performed for various geographically diverse P. falciparum isolates. The complete pfron4 nucleotide sequences from P. falciparum 3D7 (presumed African origin; (Ponnudurai et al., 1982); extracted from www.plasmodb.org and reannotated herein), HB3 (Honduras; (Walliker et al., 1987); http:// www.broad.mit.edu/) and Dd2 (Indochina; (Wellems et al., 1990); http://www.broad.mit.edu/) were extracted from their respective databases. Only partial pfron4 sequences currently exist for additional isolates, such as K1 and 7G8, on the Broad Institute website. The nucleotide and translated protein sequences of these isolates were aligned and are presented in Supplementary Figs. 1 and 2. Extremely limited sequence diversity was observed at the nucleotide (Supp. Fig. 1) and amino acid levels (Supp. Fig. 2), with only one amino acid difference being observed across the entire 1201 residues of PfRON4, which is located at residue 95 (Dd2 and HB3, Aparagine; 3D7, Isoleucine).
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(Fig. 2A) that are rich in charged amino acid residues, including glutamic acid, asparagine and glutamine. The anti-PfRON4 antisera were also used in co-localisation indirect immunofluorescence assays (IFAs) of schizont-infected red blood cells (Fig. 2C) with antisera specific for the other well characterised rhoptry proteins RAMA and Pf34 (Proellocks et al., 2007; Topolska et al., 2004). In these images, the labelling pattern observed with the anti-PfRON4 antisera demonstrated the classical punctate labelling pattern that is common to other rhoptry-located proteins (Proellocks et al., 2007; Topolska et al., 2004), and indeed demonstrated co-localisation to the rhoptries with both RAMA and Pf34. As expected, IFAs performed with antisera against the microneme proteins PfAMA1 and EBA-175 failed to show co-localisation with PfRON4 (data not shown). Previous studies in T. gondii have demonstrated that TgAMA1 and TgRON4 are found in the tight junction protein complex (Alexander et al., 2005), as are their homologous proteins PfAMA1 and PfRON4 in P. falciparum (Alexander et al., 2006). As functional confirmation of the specificity of our polyclonal anti-PfRON4 antisera, we performed immunoprecipitations on P. falciparum 3D7 parasite lysate. In these experiments, parasite lysate was immunoprecipitated with anti-PfRON4 F1 sera and samples immunoblotted with monoclonal anti-PfAMA1 1F9 (Coley et al., 2001) or monoclonal anti-PfRON4 antibodies (Baum et al., 2008; Richard et al., 2009). The reactive epitope for mAb 1F9 has been mapped to a 57 residue region within domain 1 of the PfAMA1 protein sequence (Coley et al., 2007), and recognises both the full length (83 kDa) and proteolytically processed forms (66 kDa) of PfAMA1 (Coley et al., 2006). In our immunoprecipitated samples, reactive bands of the expected sizes for both full length and processed forms of PfAMA1 were detected. These bands were not detected in samples immunoprecipitated with the corresponding rabbit’s pre-immune control sera (Fig. 2D). A control immunoblot was also performed in which identical anti-PfRON4 immunoprecipitated samples were probed with a monoclonal anti-PfRON4 antibody to confirm the specificity of our rabbit anti-PfRON4 antisera for immunoprecipitating PfRON4 (Fig. 2D). 3.3. Regions of PfRON4 are highly reactive with human hyperimmune sera
3.2. pfron4 encodes PfRON4 To confirm that the pfron4 gene sequence annotated herein indeed encodes the previously described PfRON4, we generated antigen-specific polyclonal antisera for use in subsequent immunochemical assays. Two separate regions encoded by pfron4, referred to as F1 and F2 (Fig. 2A), were amplified by PCR, cloned into pGEX-KG and the purified GST-PfRON4 F1 and -PfRON4 F2 fusion proteins were used to generate polyclonal antisera. Validation of the specific reactivity of the anti-PfRON4 polyclonal antisera was performed by several independent methodologies. A time course of highly synchronised parasite lysate samples was probed by immunoblot with each anti-PfRON4 antisera. A single reactive band of approximately 250 kDa was detected in parasite samples that contained mature-stage trophozoites and schizonts or early rings (Fig. 2B). PfRON4 expression levels tapered off in late ring to early trophozoite stage parasites, and expression was observed to re-commence again in late trophozoites. These data are in accord with the PfRON4 immunoblot data presented by Alexander et al. (2006) and recent global gene expression arrays (Ben Mamoun et al., 2001; Bozdech et al., 2003; Le Roch et al., 2004). The observed molecular mass of PfRON4 (250 kDa) when resolved by SDS–PAGE is greater than that computationally estimated (136 kDa). This discrepancy can most likely be accounted for by the presence of two extensive peptide repeats regions
GST-PfRON4 sub-fragment fusion proteins that together span the entire length of PfRON4 were proteins expressed and purified (Fig. 3B). Immunoblot detection using polyclonal anti-GST antisera was performed on all GST fusion proteins and confirmed the presence of the GST fusion tag (data not shown). In order to investigate whether PfRON4 is able to elicit an immune response during natural human malaria infections, these GST-PfRON4 fusion proteins, along with a GST control, were immunoblotted with pooled human hyperimmune sera obtained from malaria endemic regions of Vietnam (Wang et al., 2001) and Papua New Guinea (PNG; Marshall et al., 1997). For both hyperimmune sera samples, multiple reactive regions of PfRON4 protein sequence were identified. F1, F2, F4 and F6 were consistently the most reactive regions of PfRON4 detected. Low levels of reactivity against F3 (full length protein at 100 kDa) were only detected when immunoblots were massively overexposed (data not shown). Interestingly, PfRON4 F5 (full length protein at 40 kDa) was not discernibly reactive with either hyperimmune sera. The GST control was only recognised by either hyperimmune sera upon massive overexposure of immunoblots. Taken together, these data identify PfRON4 as a rhoptry protein that is able to elicit a specific immune response in natural human malaria infections, and that specific sub-regions of PfRON4 are more highly recognised than others by human hyperimmune sera.
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Fig. 2. Functional validation of anti-PfRON4 antisera. (A) Schematic representation of the full length PfRON4 protein, showing the relative locations of the intron–exon boundaries (splice sites) and peptide repeat regions (shown shaded). The relative locations of the peptide regions (F1 and F2) used to raise polyclonal sera are also indicated. Residues numbers are shown under the schematic. (B) Immunoblots using anti-PfRON4 F1 and anti-PfRON4 F2 antisera. Synchronised cultures were sampled at regular intervals across the 48 h asexual lifecycle. The lanes were loaded with approximately 1 107 parasites and are predominantly: (1) late trophozoites/schizonts; (2) schizonts/ early rings; (3) early/late rings; and (4) late rings/early trophozoites. Representative anti-PfRON4 F1 and anti-PfRON4 F2 immunoblots are shown. A duplicate immunoblot was probed with anti-GRP(BiP) antisera ((Kumar et al., 1991) as a loading control. GRP(BiP) has previously been shown by microarray to be constitutively expressed across the 48 h cycle (Ben Mamoun et al., 2001), although slightly higher levels of expression were detected here in samples rich in mature stage parasites. (C) Co-localisation IFAs were performed on mature schizont-infected red blood cells and ruptured merozoites using our anti-PfRON4 antisera and antisera against the rhoptry marker proteins RAMA and Pf34 (Proellocks et al., 2007; Topolska et al., 2004). Co-localisation of PfRON4 with both RAMA and Pf34 is observed, with marginally better co-localisation observed with Pf34, a reported rhoptry neck protein (Proellocks et al., 2007). (D) Immunoprecipitation of PfAMA1 with PfRON4. Parasite samples immunoprecipitated with anti-PfRON4 F1 antisera were immunoblotted with monoclonal anti-PfAMA1 1F9 antibody, which is reactive with both full length PfAMA1 (F/L; 83 kDa) and its proteolytically processed form (P/P; 66 kDa) (Coley et al., 2006). Bands of the corresponding sizes (indicated by the arrows, left panel) were detected in the control parasite lysate after initial lysis (L) and non-immunoprecipitated (NI) samples, in addition to the supernatant (S) and pellet (P) fractions after initial parasite lysis and subsequent centrifugation. Both the full length and processed PfAMA1 bands were detected after immunoprecipitation (I) with anti-PfRON4 antisera, but not when the corresponding rabbit’s pre-immune (PIheaded samples) control sera was used in simultaneously performed experiments. A parallel control immunoblot was performed in which identical immunoprecipitated samples were probed with a monoclonal anti-PfRON4 antibody (Baum et al., 2008; Richard et al., 2009). PfRON4 proteins (arrow, right panel) were detected in the sample precipitated with rabbit anti-PfRON4 antisera (I, right panel) but were absent when the rabbit’s pre-immune control (PI) sera were used (I, right panel).
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Fig. 3. PfRON4 is reactive with human hyperimmune sera. (A) Schematic representation of the full length PfRON4 protein, showing the relative locations of the intron–exon boundaries (splice sites) and the peptide repeat regions (shown shaded). The relative locations of the various recombinant PfRON4 sub-fragment proteins are also indicated. Residues numbers are shown under the schematic. (B) Purified GST-PfRON4 fusion proteins. Approximately 2 lg (total) of each purified protein was resolved SDS–PAGE and stained with Coomassie brilliant blue. The protein samples are GST (labelled GST), GST-PfRON4 F1 (F1), GST-PfRON4 F2 (F2), GST-PfRON4 F3 (F3), GST-PfRON4 F4 (F4), GSTPfRON4 F5 (F5) and GST-PfRON4 F6 (F6). (C) PfRON4 fusion proteins are reactive with human hyperimmune sera. Approximately 50 lg (total) of each purified protein was immunoblotted with hyperimmune sera obtained from malaria endemic regions of Vietnam and Papua New Guinea (PNG) or non-immune sera obtained from individuals in Australia not previously exposed to malaria. Representative immunoblots detected with each hyperimmune sera pool are shown. The same regions of PfRON4 were reactive, some highly, with hyperimmune sera from both Vietnam and PNG.
4. Discussion Recent investigations in Toxoplasma gondii have identified a series of rhoptry neck proteins (TgRON4, TgRON2 and TgRON5) that localise with the tight junction and are found in complex with the T. gondii Apical Membrane Protein 1 (Alexander et al., 2005; Lebrun et al., 2005). Homologues of each of these proteins have been identified in P. falciparum, and it appears that PfRON4 is similarly found in complex with PfRON2, PfRON5 and PfAMA1 (Alexander et al., 2006; Cao et al., 2008; Collins et al., 2009). Here, we report the complete pfron4 gene structure in the P. falciparum genome and show the extreme sequence conservation of pfron4/ PfRON4 amongst geographically diverse P. falciparum isolates. Our data also demonstrate that PfRON4 protein sequences are immunogenic in natural human malaria infections. Manual analysis of the nucleotide sequence from the P. falciparum genome database (http://www.plasmodb.org) previously reported to encode PfRON4 (PF11_0168; Alexander et al., 2005, 2006) indicated that it was most likely incorrectly annotated (Robert Huestis, Victorian Bioinformatics Consortium). Indeed, our RTPCR and sequencing data confirms that PfRON4 is instead encoded by a three exon gene whose sequence only partially overlaps the previously annotated PF11_0168 sequence (Fig. 1A). Polyclonal
anti-sera generated against two separate regions of the newly corrected PfRON4 sequence confirmed the expected asexual lifecycle expression profile and rhoptry location of PfRON4 in developing merozoites (Fig. 2). Immunoprecipitations using our anti-PfRON4 F1 antisera confirmed that both the full length and processed forms of PfAMA1 (83 kDa and 66 kDa, respectively; Fig. 2D) can be found in complex with PfRON4. The target epitope of mAb 1F9 has been localised to a 57 residue region in domain 1 of PfAMA1 (Coley et al., 2007). Later proteolytic processing of PfAMA1 results in the production of 48 kDa / 44 kDa PfAMA1 cleavage products (Howell et al., 2003), which correspond to PfAMA1 domains 1, 2 and 3 being cleaved from their transmembrane regions. The absence of these 48/44 kDa bands in our immunoprecipitations (Fig. 2D) suggests that a conformationally dependent binding region in PfAMA1 for the tight junction protein complex is disrupted during cleavage. During review of this manuscript, the a detailed description of the interaction between PfAMA1 and tight junction protein complex was published, demonstrating that the Tyr251 residue located in PfAMA1’s hydrophobic cleft was critically required for its interaction with the tight junction protein complex that includes PfRON4 (Collins et al., 2009). The PfRON4 protein sequence is characterised by the presence of two extensive highly charged peptide repeat regions (Fig. 2A).
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The first repeat region consists of 5 imperfect copies of QQNSNE and the second 24 imperfect copies of NEPIPIEHATTPT. It is interesting to note that the copy number and the majority of the sequence of each peptide repeat is absolutely conserved between the geographically diverse parasite isolates analysed here (the asparagine to isoleucine amino acid change at residue 95 in 3D7 occurs within the first repeat region; Supp. Fig. 2), however further investigations would be required to determine the full extent of peptide repeat conservation in a larger cohort of geographically diverse genome sequences. Such extensive peptide repeat regions are common to many P. falciparum proteins, particularly to those that are exported from the intracellular blood-stage parasite and are found in association with the red blood cell membrane skeleton, including the Mature-infected Erythrocyte Surface Antigen (MESA) and Knob-Associated Histidine-Rich Protein (KAHRP; see Cooke et al. for review (2001)). In contrast to the almost exact sequence conservation of PfRON4’s peptide repeats amongst geographically diverse isolates, the peptide repeat regions of both MESA and KAHRP show greater variance in the number of repeated elements within a particular repeat region amongst different isolates, rather than in specific nucleotide variations within an individual repeat region (Coppel, 1992; Hirawake et al., 1997; Kant and Sharma, 1996; Kun et al., 1999; Saul et al., 1992; Sharma and Kilejian, 1987; Triglia et al., 1987). Although the precise function, if any, of PfRON4’s peptide repeat regions have yet to be defined, in other P. falciparum proteins repeat regions have been suggested to act as an immunological smoke-screen to divert the human immune system (Anders, 1986; Coppel et al., 1983) and as the binding regions in the formation of protein complexes (Waller et al., 1999). Given such precise constraints in the peptide sequences of PfRON4 amongst the geographically diverse parasite isolates analysed here, it is tempting to suggest that the PfRON4 peptide repeat regions perhaps mediate an important protein interaction that is required during invasion. Obvious candidate interactions for PfRON4’s peptide repeat regions are the interactions that form the PfRON4–PfRON2–PfRON5–PfAMA1 tight junction protein complex (Alexander et al., 2006; Cao et al., 2008; Collins et al., 2009), although future protein interaction experiments using domain specific recombinant proteins would be required to confirm this. The extreme sequence conservation of PfRON4 amongst the geographically diverse P. falciparum isolates may highlight the critical requirement of PfRON4’s functional role in the successful invasion of host cells. Previous studies in T. gondii have shown that TgRON4 is apparently refractory to deletion (Alexander et al., 2005) and our own ongoing studies have so far failed to yield a pfron4 knockout P. falciparum parasite line despite repeated attempts (Morahan et al., unpublished data). We have however been able to successfully demonstrate by PCR analysis that we can genetically target the pfron4 gene by introducing a Haemagglutinin (HA) tag onto its 30 end in bulk (non-cloned) parasite transfection cultures (Morahan et al., unpublished data). Our inability to genetically delete pfron4 from the P. falciparum genome, combined with its apparent high level of protein sequence conservation across geographically diverse P. falciparum isolates leads to the suggestion that PfRON4’s function in the invasion process may be critically required. In natural human infections, secretion of the rhoptry-located proteins during merozoite invasion of the red blood cell potentially results in these proteins being exposed to the human immune system and their elicitation of an antibody response. To assess if PfRON4 elicits such an immune response in natural infections, pooled sera from individuals living in malaria endemic regions of Papua New Guinea (PNG) and Vietnam were used to immunoblot the various GST-PfRON4 sub-fragment fusion proteins generated in this study. Regions F1, F2, F4 and F6 of PfRON4
were each recognised by both hyperimmune sera, with F3 reactivity only being detected upon overexposure of the immunoblot. Reduced levels of F3 reactivity most likely result from the overall low levels of full length GST-PfRON4 F3 protein (100 kDa) being successfully expressed and purified for loading into the immunoblot (due to it containing the hydrophobic signal sequence encoded by exon 1 of pfron4) rather than its lack of immune recognition resulting from a protein conformational effect, although this can not be excluded. Interestingly, PfRON4 F5 (full length protein 40 kDa) was not appreciably reactive with hyperimmune sera from either PNG or Vietnam, thus suggesting its lack of immunogenic sequences or its possible sequestration from exposure to the human immune system during parasite invasion. It is also possible that our non-refolded recombinant protein is not correctly folded and thus is not recognised by human antibodies raised against the native parasite PfRON4 protein in natural malaria infections. This is perhaps the most likely scenario given that the C-terminal sequence of PfRON4 (that encompasses both F5 and F6) is likely to be highly structured due to the presence of six cysteine residues (Supp. Fig. 2). Also, since that PfRON4 is a highly conserved and functionally important protein, it would be most interesting to assess the variance in levels of anti-PfRON4 immunoreactivity in individual Vietnam and Papua New Guinea sera samples to gain insight into PfRON4’s potential utility as a malaria vaccine candidate. We aim to expand upon our current investigations to more rigorously address this question in the future using ELISAs. The detection of anti-PfRON4 antibodies in human hyperimmune sera is suggestive that PfRON4 is functionally exposed during merozoite invasion and is stimulatory to the human immune system in natural malaria infections. This combined with our inability to genetically delete pfron4 from the genome and its high level of sequence conservation leads us to the suggestion that PfRON4 may be suitable for further investigation as a potential malaria vaccine candidate. Two of the most highly validated proteins for inclusion in a malaria vaccine, PfAMA1 and Merozoite Surface Protein 1 19 kDa region (MSP1 19 kDa) are functionally important merozoite proteins that are required for invasion (see Cowman and Crabb for review (2006)). Two recent investigations have cast doubt on the validity of PfRON4 as a vaccine candidate. In those studies, immunisation with a recombinant his-tagged C-terminal region of P. yoelli RON4 (PyRON4) failed to show protection from lethal infection in mouse immunisation/parasite challenge experiments (Narum et al., 2008) and similarly, anti-TgRON4 antibodies failed to inhibit T. gondii invading HFF cells (Alexander et al., 2005). However, in the former study, the data were derived from the use of a protein (which contains the six C-terminal cysteine residues that are also equivalently located in PfRON4; see Supp. Fig. 2) expressed in a eukaryotic Pichia pastoris system but was not subjected to a protein re-folding protocol. In the later study, only a portion of TgRON4 (residues 85–983; Bradley et al., 2005) was used to raise the antibodies. It is therefore unlikely that both studies yield an accurate estimate of the potential of PfRON4 as a malaria vaccine candidate. To more thoroughly determine if anti-PfRON4 antibodies can mediate protection, we aim to perform future mouse immunisation/parasite challenge investigations using recombinant full length PfRON4 (or sub-fragment proteins that together span the entire protein sequence) that have been purified and re-folded. Ideally, data derived from these additional assays would need to be combined with that from parallel studies assessing anti-PfRON4 antisera in in vitro parasite growth inhibition assays and physiologically relevant antibody-dependent cellular inhibition assays (ADCI; Druilhe and Bouharoun-Tayoun, 2002) to more accurately determine if PfRON4 is indeed a potential malaria vaccine candidate protein.
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Plasmodium falciparum: Genetic and immunogenic characterisation of the rhoptry neck protein PfRON4 Belinda J. Morahan a,1, Georgina B. Sallmann a, Robert Huestis a,b, Valentina Dubljevic a, Karena L. Waller a,* a b
Department of Microbiology, Monash University, Clayton, Vic. 3800, Australia Victorian Bioinformatics Consortium, Monash University, Clayton, Vic. 3800, Australia
a r t i c l e
i n f o
Article history: Received 19 January 2009 Received in revised form 7 April 2009 Accepted 29 April 2009 Available online 12 May 2009 Keywords: PfRON4 Malaria Plasmodium falciparum Rhoptry
a b s t r a c t The Apicomplexan parasites Toxoplasma and Plasmodium, respectively, cause toxoplasmosis and malaria in humans and although they invade different host cells they share largely conserved invasion mechanisms. Plasmodium falciparum merozoite invasion of red blood cells results from a series of co-ordinated events that comprise attachment of the merozoite, its re-orientation, release of the contents of the invasion-related apical organelles (the rhoptries and micronemes) followed by active propulsion of the merozoite into the cell via an actin-myosin motor. During this process, a tight junction between the parasite and red blood cell plasma membranes is formed and recent studies have identified rhoptry neck proteins, including PfRON4, that are specifically associated with the tight junction during invasion. Here, we report the structure of the gene that encodes PfRON4 and its apparent limited diversity amongst geographically diverse P. falciparum isolates. We also report that PfRON4 protein sequences elicit immunogenic responses in natural human malaria infections. Ó 2009 Elsevier Inc. All rights reserved.
1. Introduction The obligate intracellular protozoan parasites Toxoplasma and Plasmodium are both members of the Apicomplexan family and are the respective causative agents of the human parasitic diseases toxoplasmosis and malaria. Common to the invasive forms of all Apicomplexan parasites are a set of apically located secretory organelles, the rhoptries, micronemes and dense granules, which secrete numerous proteins, some of which are critically required for invasion of the target host cell. Although Toxoplasma and Plasmodium ultimately invade different cell types, they share a commonality in invasion mechanisms. For both parasites, invasion results from a series of co-ordinated events that comprise attachment of the invasive parasite to the host cell, its re-orientation and release of the contents of the apical organelles (rhoptries and micronemes) followed by active propulsion of the parasite into the host cell via an actin-myosin motor. During invasion, the irreAbbreviations: TgRON4, Toxoplasma gondii rhoptry neck protein 4; PfAMA1, Plasmodium falciparum apical membrane antigen 1; PfRON4, Plasmodium falciparum rhoptry neck protein 4; IFA, indirect immunofluorescence assay; GST, glutathione Stransferase; DAPI, 40 ,6-diamidino-2-phenylinodole dihydrochloride; PBS, phosphate buffered saline. * Corresponding author. Fax: +61 3 9902 9222. E-mail address: [email protected] (K.L. Waller). 1 Present address: Department of Biology, Loyola University, Chicago IL, USA. 0014-4894/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2009.04.013
versible juncture formed between the plasma membranes of the invading parasite and the host cell is called the tight junction, and results from a series of protein interactions between parasite ligands and their specific receptor molecules on the host cell surface. As invasion proceeds, the tight junction translocates along the sides of the invading parasite until the invasion process eventually terminates when the parasite is fully engulfed within a parasitophorous vacuole in the host cell (see Cowman and Crabb for recent review (2006)). Studies in Toxoplasma gondii have begun to tease apart the parasite proteins associated with the tight junction during invasion of host cells, one of which is TgRON4. TgRON4 appears to be refractory to genetic deletion and localises to the neck region of the rhoptries in non-invading tachyzoites, but in invading tachyzoites TgRON4 is secreted from the rhoptries and localises with the tight junction as it translocates along the sides of the invading parasite (Alexander et al., 2005; Lebrun et al., 2005). TgRON4 has also been shown to form a protein complex consisting of two other rhoptry neck proteins, TgRON2 and TgRON5, as well as the microneme-secreted and proteolytically processed integral membrane protein that is critically required for invasion, T. gondii Apical Membrane Antigen 1 (TgAMA1) (Alexander et al., 2005; Mital et al., 2005). Highlighting the significant level of conservation in parasite invasion mechanisms between T. gondii and P. falciparum, the P. falciparum homologues of each TgRON have recently been identified
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(Alexander et al., 2006; Bradley et al., 2005). PfRON4 has been shown to localise to the neck region of rhoptries in merozoites (Alexander et al., 2006) and is similarly found in complex with the leading malaria vaccine candidate PfAMA1 (Alexander et al., 2006), PfRON2 and PfRON5 (Cao et al., 2008; Collins et al., 2009). For both T. gondii and P. falciparum, the precise function of the RON4–AMA1 protein complex has yet to be delineated. In the P. falciparum genome database (www.plasmodb.org), the gene that encodes PfRON4 is incorrectly annotated. Here, we describe the correct structure of the gene that encodes PfRON4 and probe the level of conservation of PfRON4 sequences derived from geographically diverse P. falciparum isolates. We also investigate the immunogenicity of regions of PfRON4 with hyperimmune sera obtained from individuals living in malaria endemic regions of the world.
Homologous DNA sequences to pfron4 were extracted using BLAST searches from the genome database that is available online at the Broad Institute (http://www.broad.mit.edu/). Complete pfron4 nucleotide sequences were extracted for the P. falciparum isolates HB3 (Honduras; Walliker et al., 1987) and Dd2 (Indochina; Wellems et al., 1990). The pfron4 sequence for 3D7 (a clonal line derived from NF54 and of presumed African origin; (Ponnudurai et al., 1982) was derived by our manual re-annotation of sequence available from http://www.plasmodb.org. ClustalW nucleotide and protein sequence alignments were performed using software available through the European Bioinformatics Institute website (http:// www.ebi.ac.uk/Tools/clustalw/). 2.3. GST-PfRON4 fusion protein expression and purification
Plasmodium falciparum 3D7 parasites were maintained by in vitro culture using standard culture conditions (Trager and Jensen, 1976) and supplemented with 0.5% (w/v) AlbumaxII (Invitrogen). Parasitaemias were determined by microscopic examination of Giemsa stained thin blood smears. Highly synchronised parasite cultures were obtained by sorbitol synchronisation methods (Lambros and Vanderberg, 1979).
Recombinant glutathione S-transferase (GST) fusions proteins of regions of PfRON4 were generated. Six overlapping regions of pfron4, referred to as F1 through F6 (Fig. 3A), were amplified by PCR using specific primers (Table 1) and P. falciparum 3D7 cDNA as the template. The resultant DNA fragments were cloned into pGEX-KG (Guan and Dixon, 1991) and the encoded GST fusion proteins were expressed and purified using standard affinity chromatography (Bennett et al., 1997). The majority of the fusion proteins generated, excepting fragments F1 and F3, possessed a small overlap of 69 residues at the N- or C-terminus of each PfRON4 protein sequence resulting from overlap of the amplifying adjacent primer sequences.
2.2. PCR amplification, cloning and sequence analysis of pfron4
2.4. Antisera production and immunoblotting
Specific oligonucleotide primers (Table 1) were used to amplify full length pfron4 from P. falciparum 3D7 cDNA. Manual bioinformatic analyses performed by Robert Huestis (Victorian Bioinformatics Consortium) of the PF11_0168 nucleotide sequence (www.plasmodb.org) indicated it most likely contained an incorrect gene annotation. Our re-annotated three exon pfron4 gene structure (Fig. 1A) was confirmed by RT-PCR (Superscript II Kit, Invitrogen) using total RNA extracted with TrizolÒ (Invitrogen) from asynchronous parasite cultures. RT-PCR products were amplified using the Expand High Fidelity PCR System (Roche) from samples treated with or without Reverse Transcriptase (RT+ and RT, respectively). The amplified RT-PCR products were resolved by agarose gel electrophoresis in 1% (w/v) gels and then cloned using the pTOPO TA Cloning Kit for Sequencing (Invitrogen). Both the sense and antisense nucleotide sequences of two independent pfron4 cDNA clones were determined. Sequence analyses and alignments were performed using SeqManTM II software (DNAStar). The re-annotated and RT-PCR confirmed gene sequence of pfron4 has been reported to www.plasmodb.org.
Rabbit polyclonal antibodies were generated against the GSTPfRON4 fusion proteins F1 (amino acid residues 42–400; Proellocks et al., 2009) and F2 (amino acid residues 583–767), using previously described methods (Proellocks et al., 2009). Purified recombinant proteins and parasite lysates were resolved by denaturing SDS–PAGE in 12% (w/v) polyacrylamide gels and transferred to polyvinylidene fluoride (PVDF) membranes (NEN). Immunoblots were probed with various primary antibodies and detected using Western Lightning Chemiluminescence Reagent Plus (PerkinElmer Life Sciences). The primary antibodies used were polyclonal rabbit anti-PfRON4 F1 and anti-PfRON4 F2 (1/500), anti-GRP(BiP) antisera (1/500; Kumar et al., 1991), rabbit anti-GST (1/500 dilution) and pooled human hyperimmune sera (1/1000) obtained from malaria endemic regions of Vietnam (Wang et al., 2001) and Papua New Guinea (Marshall et al., 1997). Pooled hyperimmune sera samples consist of pooled equal volumes of at least 18 individual sera. Human control sera were obtained from individuals living in Melbourne, Australia that had not previously been exposed to malaria.
2. Materials and methods 2.1. Parasite culture
Table 1 Oligonucleotide primers. Primer
Sequence (50 ? 30 )a
Targetb
p1 p2 p3 p4 p5 p6 p7 p8 p9 p10 p11
ccggaattctaATGTCTAGTGTTAGATTTTTTTTATG cccaagcttTTATAAATCATCAAAAATCATCTTTTC ccggaattctaAGCCATATAGAAGAACCTCAA cccaagctATGTGAATGATGATTTATATTATTAT ccggaattctaTTTCCAAACGAGGATGATAATC cccaagcttTCTTTCTCTTAAAGATACATGCA ccggaattctaAATAATATAAATCATCATTCACATG cccaagcttATGATTATCATCCTCGTTTGG ccggaattctaTTGCATGTATCTTTAAGAGAAAG cccaagcttTGCTCCATATTCATCAGGAGC ccggaattcGGTTAGCTCCTGATGAATATGG
pfron4 pfron4 pfron4 pfron4 pfron4 pfron4 pfron4 pfron4 pfron4 pfron4 pfron4
a b
Sequences shown in upper case are gene specific. Underlined sequences indicate enzyme restriction sites. + and indicate sense and anti-sense strand sequences, respectively.
full length (+) and pfron4 F3 (+) full length () and pfron4 F6 () F1 (+) F1 () and pfron4 F3 () F2 (+) F2 () F4 (+) F4 () F5 (+) F5 () F6 (+)
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Fig. 1. Genomic structure of pfron4. (A) Schematic representation of the annotated, predicted and confirmed pfron4 genomic locus (exon sequences are drawn to scale). The gene sequence PF11_0168 (www.plasmodb.org) was reported as encoding PfRON4 (Alexander et al., 2005; Lebrun et al., 2005), and as such, is annotated to possess three exons in a tandem array; exon one (5471 bp) is located 999 bp upstream of exon 2 (81 bp), and exon 3 (3346 bp) is located a further 201 bp downstream. Closer examination of the genomic nucleotide sequence data indicated a likely mis-annotation. Manual re-annotation predicted that PfRON4 was instead encoded by a gene possessing three exons, in which the first exon (76 bp) was located 201 bp upstream of the second exon (3340 bp) with the third exon (190 bp) being located a further 129 bp downstream. In this prediction, the larger 5471 bp exon was excluded from the PfRON4 coding sequence. The relative locations of oligonucleotide primers used in RT-PCR are shown, along with the expected sizes of the amplified DNA products. (B) Confirmation of the predicted pfron4 genomic structure by RT-PCR. An approximately 3.6 kb cDNA fragment (indicated by the asterisk) was amplified from samples treated with Reverse Transcriptase (RT+) but not in non-treated samples (RT).
2.5. Indirect immunofluorescence assay (IFA) Thin blood smears were prepared from cultures rich in schizont stage parasites and fixed with acetone/methanol (9:1). IFAs were preformed using previously described methods (Waller et al., 2008). Cells were visualized by wide field fluorescent microscopy using an Olympus BX51 system microscope and an Olympus DP70 microscope digital camera. Images were captured using DP Controller and DPManager software (Olympus Optical Co.). The primary antibodies were: rabbit anti-PfRON4 F2 (1/500), rabbit anti-RAMA Fragment D (1/500; Topolska et al., 2004), mouse anti-Pf34 Fragment C (1/500; Proellocks et al., 2007), rat anti-EBA-175 region VI (1/500; Sim et al., 1990) and monoclonal anti-PfAMA1 1F9 (1/500; Coley et al., 2001). The secondary antibodies were: goat anti-rabbit, goat anti-rat and goat anti-mouse AlexaFluorÒ488 (Green) or AlexaFluorÒ568 (Red) conjugate antibodies (1/500; Molecular Probes). 2.6. Immunoprecipitation Immunoprecipitations of parasite material were performed using a methodology adapted from Cooke et al. (2006). Immunoprecipitated protein complexes were resolved by denaturing SDS–PAGE in 12% (v/v) acrylamide gels before immunoblotting with monoclonal anti-PfAMA1 1F9 antibody (1/1000; Coley et al., 2001) or mono-
clonal anti-PfRON4 antibody (1/500; Baum et al., 2008; Richard et al., 2009).
3. Results 3.1. pfron4 gene structure and sequence Previous reports have identified PF11_0168 as the gene sequence that encodes the rhoptry neck protein PfRON4 (Alexander et al., 2005; Lebrun et al., 2005). PF11_0168, as originally annotated in the P. falciparum genome database (www.plasmodb.org), consisted of three exons in a tandem array (Fig. 1A). Manual bioinformatic analysis of the putative intron/ exon boundaries proposed in this PF11_0168 sequence indicated that the sequence had most likely been incorrectly annotated. Manual re-annotation of this genomic locus suggested that although PfRON4 was indeed encoded by a gene possessing three exons in a tandem array, it appeared to exclude the larger 5471 bp sequence and instead include a previously unannotated downstream exon. Thus, we hypothesised that pfron4 was encoded by a first exon (76 bp) located 201 bp upstream of the second exon (3340 bp) with the third exon (190 bp) being located a further 129 bp downstream (Fig. 1A).
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In order to confirm that our manually re-annotated gene sequence encodes PfRON4, we performed RT-PCR on total RNA extracted from asynchronous parasite cultures. Specific primers (Table 1) were designed with reference to our predicted gene annotation to amplify the full length cDNA, which if our predicted exon model was correct, was expected to result in a 3606 bp spliced cDNA RT-PCR product (Fig. 1A). An approximately 3.6 kb product was successfully amplified by RT-PCR in RT+ samples. No amplification products were observed in RT- samples (Fig. 1B). The complete nucleotide sequences of two independent cloned pfron4 cDNAs were determined and compared to the reported sequence of PF11_0168 from the genome database (www.plasmodb.org). These comparisons demonstrated significant homology (data not shown) to the P. falciparum genome database’s PF11_0168 exon 2 and exon 3 sequences, but not to exon 1 (5471 bp exon; Fig. 1A). Instead, the nucleotide sequence obtained from the 30 end of both the cloned cDNA products was homologous to a previously unannotated exon located downstream of PF11_0168 exon 3. Thus, we have shown that PfRON4 is encoded by a three exon gene (Fig. 1A) that is only partially encoded by the sequence reported as PF11_0168 in the genome database (www.plasmodb.org). ClustalW alignments of the pfron4 sequence data extracted from the Broad Institute genome database (http://www.broad.mit.edu/) or www.plasmodb.org were performed for various geographically diverse P. falciparum isolates. The complete pfron4 nucleotide sequences from P. falciparum 3D7 (presumed African origin; (Ponnudurai et al., 1982); extracted from www.plasmodb.org and reannotated herein), HB3 (Honduras; (Walliker et al., 1987); http:// www.broad.mit.edu/) and Dd2 (Indochina; (Wellems et al., 1990); http://www.broad.mit.edu/) were extracted from their respective databases. Only partial pfron4 sequences currently exist for additional isolates, such as K1 and 7G8, on the Broad Institute website. The nucleotide and translated protein sequences of these isolates were aligned and are presented in Supplementary Figs. 1 and 2. Extremely limited sequence diversity was observed at the nucleotide (Supp. Fig. 1) and amino acid levels (Supp. Fig. 2), with only one amino acid difference being observed across the entire 1201 residues of PfRON4, which is located at residue 95 (Dd2 and HB3, Aparagine; 3D7, Isoleucine).
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(Fig. 2A) that are rich in charged amino acid residues, including glutamic acid, asparagine and glutamine. The anti-PfRON4 antisera were also used in co-localisation indirect immunofluorescence assays (IFAs) of schizont-infected red blood cells (Fig. 2C) with antisera specific for the other well characterised rhoptry proteins RAMA and Pf34 (Proellocks et al., 2007; Topolska et al., 2004). In these images, the labelling pattern observed with the anti-PfRON4 antisera demonstrated the classical punctate labelling pattern that is common to other rhoptry-located proteins (Proellocks et al., 2007; Topolska et al., 2004), and indeed demonstrated co-localisation to the rhoptries with both RAMA and Pf34. As expected, IFAs performed with antisera against the microneme proteins PfAMA1 and EBA-175 failed to show co-localisation with PfRON4 (data not shown). Previous studies in T. gondii have demonstrated that TgAMA1 and TgRON4 are found in the tight junction protein complex (Alexander et al., 2005), as are their homologous proteins PfAMA1 and PfRON4 in P. falciparum (Alexander et al., 2006). As functional confirmation of the specificity of our polyclonal anti-PfRON4 antisera, we performed immunoprecipitations on P. falciparum 3D7 parasite lysate. In these experiments, parasite lysate was immunoprecipitated with anti-PfRON4 F1 sera and samples immunoblotted with monoclonal anti-PfAMA1 1F9 (Coley et al., 2001) or monoclonal anti-PfRON4 antibodies (Baum et al., 2008; Richard et al., 2009). The reactive epitope for mAb 1F9 has been mapped to a 57 residue region within domain 1 of the PfAMA1 protein sequence (Coley et al., 2007), and recognises both the full length (83 kDa) and proteolytically processed forms (66 kDa) of PfAMA1 (Coley et al., 2006). In our immunoprecipitated samples, reactive bands of the expected sizes for both full length and processed forms of PfAMA1 were detected. These bands were not detected in samples immunoprecipitated with the corresponding rabbit’s pre-immune control sera (Fig. 2D). A control immunoblot was also performed in which identical anti-PfRON4 immunoprecipitated samples were probed with a monoclonal anti-PfRON4 antibody to confirm the specificity of our rabbit anti-PfRON4 antisera for immunoprecipitating PfRON4 (Fig. 2D). 3.3. Regions of PfRON4 are highly reactive with human hyperimmune sera
3.2. pfron4 encodes PfRON4 To confirm that the pfron4 gene sequence annotated herein indeed encodes the previously described PfRON4, we generated antigen-specific polyclonal antisera for use in subsequent immunochemical assays. Two separate regions encoded by pfron4, referred to as F1 and F2 (Fig. 2A), were amplified by PCR, cloned into pGEX-KG and the purified GST-PfRON4 F1 and -PfRON4 F2 fusion proteins were used to generate polyclonal antisera. Validation of the specific reactivity of the anti-PfRON4 polyclonal antisera was performed by several independent methodologies. A time course of highly synchronised parasite lysate samples was probed by immunoblot with each anti-PfRON4 antisera. A single reactive band of approximately 250 kDa was detected in parasite samples that contained mature-stage trophozoites and schizonts or early rings (Fig. 2B). PfRON4 expression levels tapered off in late ring to early trophozoite stage parasites, and expression was observed to re-commence again in late trophozoites. These data are in accord with the PfRON4 immunoblot data presented by Alexander et al. (2006) and recent global gene expression arrays (Ben Mamoun et al., 2001; Bozdech et al., 2003; Le Roch et al., 2004). The observed molecular mass of PfRON4 (250 kDa) when resolved by SDS–PAGE is greater than that computationally estimated (136 kDa). This discrepancy can most likely be accounted for by the presence of two extensive peptide repeats regions
GST-PfRON4 sub-fragment fusion proteins that together span the entire length of PfRON4 were proteins expressed and purified (Fig. 3B). Immunoblot detection using polyclonal anti-GST antisera was performed on all GST fusion proteins and confirmed the presence of the GST fusion tag (data not shown). In order to investigate whether PfRON4 is able to elicit an immune response during natural human malaria infections, these GST-PfRON4 fusion proteins, along with a GST control, were immunoblotted with pooled human hyperimmune sera obtained from malaria endemic regions of Vietnam (Wang et al., 2001) and Papua New Guinea (PNG; Marshall et al., 1997). For both hyperimmune sera samples, multiple reactive regions of PfRON4 protein sequence were identified. F1, F2, F4 and F6 were consistently the most reactive regions of PfRON4 detected. Low levels of reactivity against F3 (full length protein at 100 kDa) were only detected when immunoblots were massively overexposed (data not shown). Interestingly, PfRON4 F5 (full length protein at 40 kDa) was not discernibly reactive with either hyperimmune sera. The GST control was only recognised by either hyperimmune sera upon massive overexposure of immunoblots. Taken together, these data identify PfRON4 as a rhoptry protein that is able to elicit a specific immune response in natural human malaria infections, and that specific sub-regions of PfRON4 are more highly recognised than others by human hyperimmune sera.
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Fig. 2. Functional validation of anti-PfRON4 antisera. (A) Schematic representation of the full length PfRON4 protein, showing the relative locations of the intron–exon boundaries (splice sites) and peptide repeat regions (shown shaded). The relative locations of the peptide regions (F1 and F2) used to raise polyclonal sera are also indicated. Residues numbers are shown under the schematic. (B) Immunoblots using anti-PfRON4 F1 and anti-PfRON4 F2 antisera. Synchronised cultures were sampled at regular intervals across the 48 h asexual lifecycle. The lanes were loaded with approximately 1 107 parasites and are predominantly: (1) late trophozoites/schizonts; (2) schizonts/ early rings; (3) early/late rings; and (4) late rings/early trophozoites. Representative anti-PfRON4 F1 and anti-PfRON4 F2 immunoblots are shown. A duplicate immunoblot was probed with anti-GRP(BiP) antisera ((Kumar et al., 1991) as a loading control. GRP(BiP) has previously been shown by microarray to be constitutively expressed across the 48 h cycle (Ben Mamoun et al., 2001), although slightly higher levels of expression were detected here in samples rich in mature stage parasites. (C) Co-localisation IFAs were performed on mature schizont-infected red blood cells and ruptured merozoites using our anti-PfRON4 antisera and antisera against the rhoptry marker proteins RAMA and Pf34 (Proellocks et al., 2007; Topolska et al., 2004). Co-localisation of PfRON4 with both RAMA and Pf34 is observed, with marginally better co-localisation observed with Pf34, a reported rhoptry neck protein (Proellocks et al., 2007). (D) Immunoprecipitation of PfAMA1 with PfRON4. Parasite samples immunoprecipitated with anti-PfRON4 F1 antisera were immunoblotted with monoclonal anti-PfAMA1 1F9 antibody, which is reactive with both full length PfAMA1 (F/L; 83 kDa) and its proteolytically processed form (P/P; 66 kDa) (Coley et al., 2006). Bands of the corresponding sizes (indicated by the arrows, left panel) were detected in the control parasite lysate after initial lysis (L) and non-immunoprecipitated (NI) samples, in addition to the supernatant (S) and pellet (P) fractions after initial parasite lysis and subsequent centrifugation. Both the full length and processed PfAMA1 bands were detected after immunoprecipitation (I) with anti-PfRON4 antisera, but not when the corresponding rabbit’s pre-immune (PIheaded samples) control sera was used in simultaneously performed experiments. A parallel control immunoblot was performed in which identical immunoprecipitated samples were probed with a monoclonal anti-PfRON4 antibody (Baum et al., 2008; Richard et al., 2009). PfRON4 proteins (arrow, right panel) were detected in the sample precipitated with rabbit anti-PfRON4 antisera (I, right panel) but were absent when the rabbit’s pre-immune control (PI) sera were used (I, right panel).
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Fig. 3. PfRON4 is reactive with human hyperimmune sera. (A) Schematic representation of the full length PfRON4 protein, showing the relative locations of the intron–exon boundaries (splice sites) and the peptide repeat regions (shown shaded). The relative locations of the various recombinant PfRON4 sub-fragment proteins are also indicated. Residues numbers are shown under the schematic. (B) Purified GST-PfRON4 fusion proteins. Approximately 2 lg (total) of each purified protein was resolved SDS–PAGE and stained with Coomassie brilliant blue. The protein samples are GST (labelled GST), GST-PfRON4 F1 (F1), GST-PfRON4 F2 (F2), GST-PfRON4 F3 (F3), GST-PfRON4 F4 (F4), GSTPfRON4 F5 (F5) and GST-PfRON4 F6 (F6). (C) PfRON4 fusion proteins are reactive with human hyperimmune sera. Approximately 50 lg (total) of each purified protein was immunoblotted with hyperimmune sera obtained from malaria endemic regions of Vietnam and Papua New Guinea (PNG) or non-immune sera obtained from individuals in Australia not previously exposed to malaria. Representative immunoblots detected with each hyperimmune sera pool are shown. The same regions of PfRON4 were reactive, some highly, with hyperimmune sera from both Vietnam and PNG.
4. Discussion Recent investigations in Toxoplasma gondii have identified a series of rhoptry neck proteins (TgRON4, TgRON2 and TgRON5) that localise with the tight junction and are found in complex with the T. gondii Apical Membrane Protein 1 (Alexander et al., 2005; Lebrun et al., 2005). Homologues of each of these proteins have been identified in P. falciparum, and it appears that PfRON4 is similarly found in complex with PfRON2, PfRON5 and PfAMA1 (Alexander et al., 2006; Cao et al., 2008; Collins et al., 2009). Here, we report the complete pfron4 gene structure in the P. falciparum genome and show the extreme sequence conservation of pfron4/ PfRON4 amongst geographically diverse P. falciparum isolates. Our data also demonstrate that PfRON4 protein sequences are immunogenic in natural human malaria infections. Manual analysis of the nucleotide sequence from the P. falciparum genome database (http://www.plasmodb.org) previously reported to encode PfRON4 (PF11_0168; Alexander et al., 2005, 2006) indicated that it was most likely incorrectly annotated (Robert Huestis, Victorian Bioinformatics Consortium). Indeed, our RTPCR and sequencing data confirms that PfRON4 is instead encoded by a three exon gene whose sequence only partially overlaps the previously annotated PF11_0168 sequence (Fig. 1A). Polyclonal
anti-sera generated against two separate regions of the newly corrected PfRON4 sequence confirmed the expected asexual lifecycle expression profile and rhoptry location of PfRON4 in developing merozoites (Fig. 2). Immunoprecipitations using our anti-PfRON4 F1 antisera confirmed that both the full length and processed forms of PfAMA1 (83 kDa and 66 kDa, respectively; Fig. 2D) can be found in complex with PfRON4. The target epitope of mAb 1F9 has been localised to a 57 residue region in domain 1 of PfAMA1 (Coley et al., 2007). Later proteolytic processing of PfAMA1 results in the production of 48 kDa / 44 kDa PfAMA1 cleavage products (Howell et al., 2003), which correspond to PfAMA1 domains 1, 2 and 3 being cleaved from their transmembrane regions. The absence of these 48/44 kDa bands in our immunoprecipitations (Fig. 2D) suggests that a conformationally dependent binding region in PfAMA1 for the tight junction protein complex is disrupted during cleavage. During review of this manuscript, the a detailed description of the interaction between PfAMA1 and tight junction protein complex was published, demonstrating that the Tyr251 residue located in PfAMA1’s hydrophobic cleft was critically required for its interaction with the tight junction protein complex that includes PfRON4 (Collins et al., 2009). The PfRON4 protein sequence is characterised by the presence of two extensive highly charged peptide repeat regions (Fig. 2A).
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The first repeat region consists of 5 imperfect copies of QQNSNE and the second 24 imperfect copies of NEPIPIEHATTPT. It is interesting to note that the copy number and the majority of the sequence of each peptide repeat is absolutely conserved between the geographically diverse parasite isolates analysed here (the asparagine to isoleucine amino acid change at residue 95 in 3D7 occurs within the first repeat region; Supp. Fig. 2), however further investigations would be required to determine the full extent of peptide repeat conservation in a larger cohort of geographically diverse genome sequences. Such extensive peptide repeat regions are common to many P. falciparum proteins, particularly to those that are exported from the intracellular blood-stage parasite and are found in association with the red blood cell membrane skeleton, including the Mature-infected Erythrocyte Surface Antigen (MESA) and Knob-Associated Histidine-Rich Protein (KAHRP; see Cooke et al. for review (2001)). In contrast to the almost exact sequence conservation of PfRON4’s peptide repeats amongst geographically diverse isolates, the peptide repeat regions of both MESA and KAHRP show greater variance in the number of repeated elements within a particular repeat region amongst different isolates, rather than in specific nucleotide variations within an individual repeat region (Coppel, 1992; Hirawake et al., 1997; Kant and Sharma, 1996; Kun et al., 1999; Saul et al., 1992; Sharma and Kilejian, 1987; Triglia et al., 1987). Although the precise function, if any, of PfRON4’s peptide repeat regions have yet to be defined, in other P. falciparum proteins repeat regions have been suggested to act as an immunological smoke-screen to divert the human immune system (Anders, 1986; Coppel et al., 1983) and as the binding regions in the formation of protein complexes (Waller et al., 1999). Given such precise constraints in the peptide sequences of PfRON4 amongst the geographically diverse parasite isolates analysed here, it is tempting to suggest that the PfRON4 peptide repeat regions perhaps mediate an important protein interaction that is required during invasion. Obvious candidate interactions for PfRON4’s peptide repeat regions are the interactions that form the PfRON4–PfRON2–PfRON5–PfAMA1 tight junction protein complex (Alexander et al., 2006; Cao et al., 2008; Collins et al., 2009), although future protein interaction experiments using domain specific recombinant proteins would be required to confirm this. The extreme sequence conservation of PfRON4 amongst the geographically diverse P. falciparum isolates may highlight the critical requirement of PfRON4’s functional role in the successful invasion of host cells. Previous studies in T. gondii have shown that TgRON4 is apparently refractory to deletion (Alexander et al., 2005) and our own ongoing studies have so far failed to yield a pfron4 knockout P. falciparum parasite line despite repeated attempts (Morahan et al., unpublished data). We have however been able to successfully demonstrate by PCR analysis that we can genetically target the pfron4 gene by introducing a Haemagglutinin (HA) tag onto its 30 end in bulk (non-cloned) parasite transfection cultures (Morahan et al., unpublished data). Our inability to genetically delete pfron4 from the P. falciparum genome, combined with its apparent high level of protein sequence conservation across geographically diverse P. falciparum isolates leads to the suggestion that PfRON4’s function in the invasion process may be critically required. In natural human infections, secretion of the rhoptry-located proteins during merozoite invasion of the red blood cell potentially results in these proteins being exposed to the human immune system and their elicitation of an antibody response. To assess if PfRON4 elicits such an immune response in natural infections, pooled sera from individuals living in malaria endemic regions of Papua New Guinea (PNG) and Vietnam were used to immunoblot the various GST-PfRON4 sub-fragment fusion proteins generated in this study. Regions F1, F2, F4 and F6 of PfRON4
were each recognised by both hyperimmune sera, with F3 reactivity only being detected upon overexposure of the immunoblot. Reduced levels of F3 reactivity most likely result from the overall low levels of full length GST-PfRON4 F3 protein (100 kDa) being successfully expressed and purified for loading into the immunoblot (due to it containing the hydrophobic signal sequence encoded by exon 1 of pfron4) rather than its lack of immune recognition resulting from a protein conformational effect, although this can not be excluded. Interestingly, PfRON4 F5 (full length protein 40 kDa) was not appreciably reactive with hyperimmune sera from either PNG or Vietnam, thus suggesting its lack of immunogenic sequences or its possible sequestration from exposure to the human immune system during parasite invasion. It is also possible that our non-refolded recombinant protein is not correctly folded and thus is not recognised by human antibodies raised against the native parasite PfRON4 protein in natural malaria infections. This is perhaps the most likely scenario given that the C-terminal sequence of PfRON4 (that encompasses both F5 and F6) is likely to be highly structured due to the presence of six cysteine residues (Supp. Fig. 2). Also, since that PfRON4 is a highly conserved and functionally important protein, it would be most interesting to assess the variance in levels of anti-PfRON4 immunoreactivity in individual Vietnam and Papua New Guinea sera samples to gain insight into PfRON4’s potential utility as a malaria vaccine candidate. We aim to expand upon our current investigations to more rigorously address this question in the future using ELISAs. The detection of anti-PfRON4 antibodies in human hyperimmune sera is suggestive that PfRON4 is functionally exposed during merozoite invasion and is stimulatory to the human immune system in natural malaria infections. This combined with our inability to genetically delete pfron4 from the genome and its high level of sequence conservation leads us to the suggestion that PfRON4 may be suitable for further investigation as a potential malaria vaccine candidate. Two of the most highly validated proteins for inclusion in a malaria vaccine, PfAMA1 and Merozoite Surface Protein 1 19 kDa region (MSP1 19 kDa) are functionally important merozoite proteins that are required for invasion (see Cowman and Crabb for review (2006)). Two recent investigations have cast doubt on the validity of PfRON4 as a vaccine candidate. In those studies, immunisation with a recombinant his-tagged C-terminal region of P. yoelli RON4 (PyRON4) failed to show protection from lethal infection in mouse immunisation/parasite challenge experiments (Narum et al., 2008) and similarly, anti-TgRON4 antibodies failed to inhibit T. gondii invading HFF cells (Alexander et al., 2005). However, in the former study, the data were derived from the use of a protein (which contains the six C-terminal cysteine residues that are also equivalently located in PfRON4; see Supp. Fig. 2) expressed in a eukaryotic Pichia pastoris system but was not subjected to a protein re-folding protocol. In the later study, only a portion of TgRON4 (residues 85–983; Bradley et al., 2005) was used to raise the antibodies. It is therefore unlikely that both studies yield an accurate estimate of the potential of PfRON4 as a malaria vaccine candidate. To more thoroughly determine if anti-PfRON4 antibodies can mediate protection, we aim to perform future mouse immunisation/parasite challenge investigations using recombinant full length PfRON4 (or sub-fragment proteins that together span the entire protein sequence) that have been purified and re-folded. Ideally, data derived from these additional assays would need to be combined with that from parallel studies assessing anti-PfRON4 antisera in in vitro parasite growth inhibition assays and physiologically relevant antibody-dependent cellular inhibition assays (ADCI; Druilhe and Bouharoun-Tayoun, 2002) to more accurately determine if PfRON4 is indeed a potential malaria vaccine candidate protein.
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